Open access

1
Serogroups and genotypes of Leptospira spp. strains from bovine aborted fetuses
2
Delooz Laurent1,2, Czaplicki Guy1, Gregoire Fabien1, Dal Pozzo Fabiana2, Pez Floriane3, Kodjo
3
Angeli4, Saegerman Claude2
4
1
Regional Association for Animal Registration and Health (ARSIA) asbl, 5590 Ciney, Belgium
5
2
Research Unit of Epidemiology and Risk Analysis applied to veterinary science (UREAR-
6
ULg), Fundamental and Applied Research for Animals & Health (FARAH) Center, Faculty of
7
Veterinary Medicine, University of Liege, 4000 Liege, Belgium
8
3
BioSellal, 69007 Lyon, France
9
4
Laboratoire des Leptospires, VetAgro Sup, Campus Vétérinaire de Lyon, 69280 Marcy-
10
l’Etoile, France
11
12
ABSTRACT
13
Leptospirosis is a global disease of animals, with potential major economic impact on livestock
14
industry and important zoonotic capacities. The disease represents a major challenge in the
15
developing countries as humans and animals frequently live in close association. The serovar
16
Hardjo of Leptospira whose primary host is cattle has been studied extensively, but few data
17
exist on other current circulating or emerging serotypes. To better understand the disease in
18
cattle and how to prevent and/or control it, it is necessary to identify the genotype and the
19
serotype of circulating Leptospira. This paper presents results of several investigations
20
performed on a historical Belgian collection of congenital jaundice in bovine aborted fetuses
21
coming from the leptospirosis emerging episode of 2014 (Delooz et al., 2015). The results
22
revealed that L. Grippotyphosa and L. Australis were the most prevalent serogroups with
23
respectively 17/42 and 13/42 positive MAT during this emerging event associated with the
24
same clinical pattern. The study also confirms that congenital jaundice is associated with L.
25
kirscheneri and L. interrogans and provides the genotyping of DNA obtained from these two
26
species.
27
Keywords: Abortion, Cattle, Icteric, Jaundice, Genotyping, Leptospira, Leptospirosis,
28
Belgium
29
30
INTRODUCTION
31
In Belgium, a national surveillance program based on the compulsory reporting of
32
abortions and subsequent analyses on their products reached several objectives including
33
official surveillance of bovine brucellosis but also the monitoring of other bovine abortive
34
diseases. Some emerging or re-emerging pathogens could be identified, including Bluetongue
35
virus serotype 8, Brucella abortus and more recently Schmallenberg virus.
36
Since July 2014, the Belgian Walloon region faced an unexpected situation with a
37
drastic increase of congenital jaundice in bovine aborted fetuses (Delooz et al., 2015). During
38
the last six years, abortions associated with jaundice had been notified but the monthly
39
incidence of cases remained stable. From July to December 2014, an increase of bovine aborted
40
fetuses with jaundice was reported by ARSIA pathologists, with a significantly higher incidence
41
than previous months and years. The standardized panel of analyses designed to extend the
42
diagnosis did not allow identifying the etiology. After additional analyzes, high levels of
43
antibodies against Leptospira serogroups Australis and Grippotyphosa were found in cows after
44
abortion of icteric fetuses. Serology performed during the emergence identified serogroups
45
without providing information on the genotype of the involved pathogenic Leptospira strains.
46
A leptospiral infection was consequently hypothezised (Delooz et al., 2015).
2
47
Leptospirosis is a transmissible disease of animals and humans caused by the spirochete
48
Leptospira. All the pathogenic leptospires were formerly classified as members of the species
49
Leptospira interrogans. However, the genus has been reorganised and pathogenic Leptospira
50
are now classified in 23 species (Adler, 2010; Levett, 2001; Morey et al., 2006) from which
51
more than 300 distinct serovars included within 24 serogroups are distinguished (Levett, 2015).
52
Laboratory diagnosis of leptospirosis can be complex and involves tests designed to detect
53
specific antibodies against Leptospira, as well as for direct isolation of leptospires, antigens
54
detection and detection of Leptospira nucleic acid in animal tissues or body fluids (OIE, 2014).
55
In cattle, the seroprevalence of the serogroup Sejroe is one of the most studied and varies
56
widely from one country to another with 33% in France (Ayral et al., 2014), 30% in the
57
Netherlands (Hartman et al., 1989) and up to 83.59% in Ireland (Ryan et al., 2012). In addition,
58
antibodies against Leptospira serovar Hardjo were found in tank milk of 9.2% bovine dairy
59
herds, with a higher incidence in the southern part of the country (Dom et al., 1991). In France,
60
microscopic agglutination test (MAT) performed on samples collected in 394 cattle herds
61
allowed to determine the distribution of the following three predominant circulating serogroups
62
(Australis, Sejroe, and Grippotyphosa) (Ayral et al., 2014). In this part of Europe, leptospirosis
63
is prevalent and may be responsible for pathological events in human (Mori et al., 2014) and
64
animal health (Delooz et al., 2015).
65
Leptospirosis is a known cause of abortions and infection can be accompanied by a wide
66
variety of clinical signs. However, fetal jaundice was not observed during experimental
67
infections (Ellis and Michma, 1977; Ellis et al., 1986; Smith et al., 1997). On the contrary based
68
on the recent field observations, the hypothesis of the association of bovine fetal jaundice with
69
leptospirosis was formulated (Delooz et al., 2015). Currently, little epidemiological information
70
exists concerning the different serovars of Leptospira circulating among cattle in Belgium.
71
While the manifestations of the disease can be very different depending on the strain and the
3
72
animal species, it is important to identify the pathogenic strain in order to tackle the best
73
prevention and control measures and thereby, prevent potential transmission to humans
74
(Evangelista and Coburn, 2010).
75
76
The aim of this work was to characterize the Leptospira infection detected in bovine
abortion cases associated with fetal jaundice which occurred in Southern Belgium in 2014.
77
78
MATERIAL AND METHODS
79
Study desing
80
In the context of the Belgian passive surveillance program for bovine brucellosis, a total
81
of 42 bovine abortion cases collected from October 2011 to December 2014 were included in
82
this study. They originated from 39 cattle farms distributed among the 5 Walloon provinces.
83
They were included in the study according to the diagnosis of congenital jaundice (N = 41) or
84
the PCR positivity against pathogenic Leptospira (N = 1).
85
Information issued from the anamnesis, such as sampling date, herd identification
86
number, cattle breed, month of pregnancy and number of parity were encoded in the laboratory
87
information management system (LIMS) or in an Access database. None of these herds
88
applied vaccination against Leptospira species and, moreover, no Leptospira vaccine has a
89
marketing authorization in Belgium. The geographical localization of each case of abortion was
90
possible using the Lambert coordinates and the Belgian cattle identification and movement
91
traceability system (SANITRACE).
92
93
Laboratory analyses
94
A standardized panel of analyses was first applied to perform the laboratory diagnosis
95
of bovine abortion on submitted fetuses. Direct and/or indirect detection of pathogens was
96
performed, including bacteria (Brucella spp., Campylobacter spp., Coxiella burnetii,
4
97
Leptospira borgpetersenii and interrogans serovar Hardjo, Listeria monocytogenes, Neospora
98
caninum, Salmonella Dublin), viruses (bluetongue virus serotype 8 (BTV-8), bovine
99
herpesvirus 1 (BoHV-1), bovine herpesvirus 4 (BoHV-4), bovine viral diarrhea virus (BVDV),
100
Schmallenberg virus), several mycotic agents, and many other opportunistic bacteria (Table I).
101
102
Microscopic agglutination tests
103
MAT was performed on the 42 maternal sera sampled on the aborted cows at the time of
104
abortion using twenty-four serovars representing 14 serogroups of pathogenic Leptospira
105
species: Icterohaemorrhagiae, Copenhageni, Australis, Bratislava, Munchen, Autumnalis, Bim,
106
Castellonis, Bataviae, Canicola, Hebdomadis, Panama, Mangus, Pomona, Mozdok, Pyrogenes,
107
Sejroe, Saxkoebing, Hardjo, Wolffi, Tarassovi, Cynopteri, Vanderhoedoni, and Grippotyphosa
108
(Table II). According to observations recorded by Chappel and collaborators in 2004, titers of
109
160 or higher were defined as positive agglutination reactions for ruminants. The end-point was
110
the highest dilution of serum in which 50% agglutination still occured.
111
112
Pathogenic Leptospira DNA detection (real-time PCR).
113
During the necropsy, a spleen, kidney, liver and placenta fragment were sampled on
114
each abortion cases, pooled and stored at -20°C. PCR analysis was performed on 26 pools of
115
organs sampled from icteric fetuses, retrieved in the historical abortion samples collection
116
described in the study design (not available for 16 other fetuses). DNA extraction was
117
performed using KingFisher
118
UK) and LSI MagVet TM Universal Isolation Kit (Life Technologies, UK) and was followed by
119
pathogenic Leptospira DNA detection using a commercial PCR test (TaqVet TM PathoLept TM,
120
Thermofisher, France) on organ pool according to the manufacturer's instructions (Levett et al.,
121
2005).
TM
Flex 96 Magnetic Particle Processors (Thermo Scientific
TM
,
5
122
The PCR reactions were performed with a Stratagene Mx3500P (Agilent Technologies,
123
USA). According to the manufacturer's instructions, a sample was considered positive with a
124
threshold cycle (= Ct) value lower than 46.
125
126
Leptospira genotyping method by multilocus sequence typing (MLST) by Next-Generation
127
Sequencing (NGS)
128
Among 10 randomly selected samples (i.e. PCR real-time positive group), genotyping was
129
realized in the Biosellal laboratory of Lyon (France) according to the consensus MLST scheme
130
developed by Boonsilp and collaborators (2013). Obtained PCR amplicons were purified using
131
1.8X Agencourt AMPure XP beads (Beckman Coulter). Qubit fluorometer 2.0 (Life
132
technologies) was used to quantitate and normalize amplicon concentrations, accounting for the
133
different amplicon fragment lengths; this was followed by equimolar pooling of all MLST loci
134
for each sample.
135
Preparation of Illumina libraries was performed with 1 ng of pooled amplicons according to the
136
Nextera XT protocol (Version January 2015) and libraries were sequenced using the MiSeq
137
Personal Sequencer (Illumina). Bio-Informatic analyses were performed using CLC Genomics
138
Workbench (CLC Bio, Qiagen, Aarhus, Denmark).
139
The combination of the sequences of these seven loci was compared with a public database, the
140
Leptospira spp. MLST Databases. This publication made use of the Leptospira MLST website
141
(http://pubmlst.org/leptospira/) developed by Keith Jolley and sited at the University of Oxford
142
(Jolley and Maiden, 2010). This is located at the Imperial College of London and was funded
143
by the Wellcome Trust using Boonsilp and collaborators (2013) protocols (MLST scheme 1) to
144
determine the species of Leptospira and serogroup. Briefly, each sequenced MLST locus for
145
one sample was assigned to allele numbers and combined into an allelic profile which is then
146
assigned to a unique sequence type (ST).
6
147
148
RESULTS
149
Among the 42 abortion cases, using the standardized panel of analyses (Table I), it was
150
possible to identify one pathogen in 11 cases, Anaplasma phagocytophilum (1), Coxiella
151
burnetii (2), Neospora caninum (1) including 7 opportunistic bacteria such as Escherichia coli
152
(5), Hafnia alvei (1), and Lactococcus lactis (1). Only one maternal serum was seropositive for
153
the Leptospira serovar Hardjo Elisa but the MAT revealed negative results for all the tested
154
serogroups including for serovar Hardjo with low titers of 40.
155
156
Microscopic agglutination tests
157
Among the 42 samples of maternal serum, 29 had a positive result with respect to at
158
least one serogroup (Table III). Among the positive samples, agglutination was observed
159
against an average of 2 and a maximum of 5 serogroups per sample. A titer of ≥ 160 was used
160
to define a seropositivity reaction, no positive results have been observed for the following 5
161
serogroups as Ballum, Bataviae, Canicola, Pomona and Tarassovi. The results revealed that
162
Leptospira serogroups Grippotyphosa and Australis were the most prevalent with respectivly
163
17/42 and 13/42 positive MAT (Table III).
164
165
Leptospira interrogans spp. (RT-PCR)
166
Of the 26 organ samples analysed, DNA of pathogenic Leptospira was detected in 21
167
cases. Among the 21 PCR positive cases, 5 sera were negative by MAT against the 24 serovars
168
(14 serogroups) tested.
169
Concerning the 5 PCR negative cases, a positive MAT was observed in 4 samples. For
170
only one sample, the PCR and the MAT were negative. The results of serology (MAT) and
171
molecular detection (real-time PCR) tests are summarized in Table III.
7
172
173
Leptospira MLST genotyping
174
Among the 10 samples, 2 were successfully amplified and sequenced for all the 7 loci.
175
For these two samples, different sequence types (ST) were obtained, the ST number 110 profile
176
for sample CI-14-061536-002 and the ST number 24 profile for sample CI-12-000889-002
177
(Table III). In the Leptospira MLST database, ST number 110 profile corresponds to
178
Leptospira kirschneri species and Grippotyphosa serogroup (Table III), whereas the ST
179
number 24 profile corresponds to Leptospira interrogans species and Australis serogroup
180
(Table III).
181
182
DISCUSSION
183
MAT and PCR results support the hypothesis that the jaundice observed in fetuses was
184
due to leptospiral infection. Furthermore, no other cause of abortion was identified despite the
185
wide range of analyzes.
186
The MAT is the serological reference test, particularly appropriate for carrying out
187
epidemiological studies, since it can be applied to sera from any animal species, and because
188
the range of antigens utilized can be expanded or decreased as required (Levett, 2004). Most
189
cases of leptospirosis are diagnosed by serology and antibodies are detectable in the blood
190
approximately 5 to 7 days after the onset of clinical signs. In our conditions, the sampling was
191
performed at the time of abortion but the presence of jaundice indicated an earlier infection that
192
could explain the relative high titer in MAT observed.
193
Australis and Grippotyphosa are identified as the two predominant serogroups in this
194
study. These results are consistent with the findings of two other recent studies, in Germany
195
concerning dogs (Mayer-Scholl et al., 2013) and in France concerning dogs and cattle (Ayral
196
et al., 2014). In France, the two predominant infecting serogroups involved in clinical bovine
8
197
and canine leptospirosis from 2008 to 2011 are also Australis and Grippotyphosa for the two
198
species.
199
On average, sera show a seropositive reaction to two serogroups with a maximum of
200
five. Indeed, MAT is a complex test to control, perform, and interpret. It is a serogroup-specific
201
assay but interpretation of the MAT is complicated by the high degree of cross-reaction that
202
occurs between different serogroups, especially in acute-phase samples. This “paradoxical”
203
reaction, in which the highest titers are detected to a serogroup unrelated to the infecting one,
204
are also common and studied (Blanco et al., 2016; Lin et al., 1997). The broad cross-reactivity
205
in the acute phase is followed by relative serogroup specificity in convalescent-phase samples
206
(Levett, 2001). Than, an average of two serogroups per sample seems relatively high compared
207
to other studies where only one serovar is highlighted. But this observation argues for
208
paradoxical reaction due to IgM during acute infection. Unfortunatly, paired sera are not
209
available to confirm the infected serogroup with certainty. Moreover, a positive result does not
210
identify with certainty the cause of abortions and it is impossible to date the infection, the
211
reaction kinetics with respect to different serovars may vary (Levett, 2001).
212
In order to ensure the involvement of bacteria in the abortive process, PCR were
213
performed and bacterial DNA was detected in the great majority of cases. Following these PCR
214
analyzes, different profiles combining PCR and MAT are observed. These profiles may depend
215
on the delay between the infection and the abortion, the immunity of the infected animals, the
216
type of serogroups concerned or the laboratory assays.
217
In total, in the great majority of cases, both analyzes provide a positive response and support
218
the diagnosis of leptospirosis.
219
Because of the difficulties associated with serological identification of leptospiral
220
isolates, there has been great interest in molecular methods for identification and subtyping
221
(Terpstra, 1992; Herrmann, 1993). The reclassification of leptospires on genotypic grounds is
9
222
taxonomically correct and provides a strong foundation for future classifications. Genotyping
223
of two species of Leptospira is a key point that allows knowing with certainty the infecting
224
species. Leptospira interrogans and Leptospira kirschneri were genotyped and have a positive
225
response to serogroup Australis and Grippotyphosa respectively. These results are consistent
226
with Leptospira MLST database.
227
During this episode in 2014, many questions arise about the almost simultaneous
228
distribution on a broad territory of a disease that does not have the dissemination power of an
229
arbovirus. The disease is maintained in nature by chronic infection of reservoir hosts. The most
230
important reservoir hosts are small mammals, which may transfer infection to domestic farm
231
animals, dogs, and humans. Different rodent species may act as reservoir of the serogroups
232
highlighted in this study (Levett, 2001). Distinct variations in reservoir hosts and the serovars
233
they carry occur throughout the world (Hartskeerl and Terpstra, 1996). Currently, the source of
234
infection remains unknown and therefore complicates the usefulness of implementation of
235
preventive measures. From the affected farms, only one case of bovine aborted fetus was
236
identified in 95% of the farms with a maximum of 3 cases in one farm (Delooz et al., 2015).
237
That allows hypothesizing that infection does not spread to the entire herd by other cattle that
238
therefore do not appear to be potential maintenance hosts.
239
The spectrum of clinical signs is extremely broad. Formerly it was considered that
240
distinct clinical syndromes were associated with specific serogroups. However, this hypothesis
241
was questioned by some authors and following studies refuted this hypothesis (Levett, 2001).
242
Grippotyphosa and Australis are the two main serogroups revealed during this emerging event
243
associated with the same clinical pattern, which joins the idea that clinical syndromes were not
244
associated with specific serogroups. However, congenital jaundice from aborted fetuses coming
245
from clinically healthy cows is a new clinical sign to add to those caused by pathogenic
246
Leptospira.
10
247
The results of this study indicated that Sejroe serogroup was rarely diagnosed and that
248
methods of the diagnosis of leptospirosis must be adapted for a better surveillance and control.
249
This finding suggests that the available bovine vaccine targeting this serogroup is capable of
250
preventing a minority of the clinical cases. Nevertheless, additional serogroups, such as
251
Grippotyphosa and Australis should be included in the vaccine to eliminate most Leptospira-
252
related diseases in cattle.
253
254
CONCLUSION
255
256
Jaundice was a known clinical sign of leptospirosis but, to our kowledge, had never been
257
diagnosed in bovine aborted fetuses coming from clinically healthy cows in the literature. This
258
work allows the association of two pathogenic Leptospira species (L. interrogans or L.
259
kirschneri) to congenital jaundice in bovine aborted fetuses. This new clinical sign should be
260
added to the clinical picture of bovine leptospirosal abortion.
261
Leptospira are often difficult to isolate from infected cattle and therefore diagnosis
262
usually depends on the detection of specific antibodies. This work showed a feasible method of
263
direct diagnostic approach under field conditions where the veterinary practitioner performs
264
samples in cattle farms.
265
Finally, despite that the sources of infection during the emergence remain unknow, this
266
study provided useful information in the knowledge of bovine leptospirosis in south part of
267
Belgium. It seems necessary to be prepared to tackle appropriate prevent and control measures
268
and to further explores the epidemiology of this disease in this region, especially in wild life.
269
270
AKNOWLEDGEMENT
11
271
We thank Magnée D. (Thermofisher, UK) to provide PCR kits. We thank the Federal Agency
272
for the Safety of the Food Chain (FAFSC) to support the costs of the basic protocol
273
corresponding to the standardized panel of analyses. The authors gratefully acknowledge their
274
veterinary colleagues from ARSIA, i.e. Christian Quinet (serology), Marc Saulmont and
275
Thierry Petitjean (autopsy and microbiology) for their technical assistance.
276
277
REFERENCES
278
Adler, B., 2010: Leptospira and Leptospirosis. Current Topics in Microbiology and
279
Immunology 387: 1–293.
280
Ayral, F. C., D. J. Bicout, H. Pereira, M. Artois, and A. Kodjo, 2014: Distribution of Leptospira
281
serogroups in cattle herds and dogs in France. The American journal of tropical
282
medicine and hygiene 91, 756-759.
283
Blanco, R. M., L. F. dos Santos, R. L. Galloway, and E. C. Romero, 2016: Is the
284
microagglutination test (MAT) good for predicting the infecting serogroup for
285
leptospirosis in Brazil? Comparative immunology, microbiology and infectious diseases
286
44, 34-36.
287
Boonsilp, S., Thaipadungpanit, J., Amornchai, P., Wuthiekanun, V., Bailey, M.S., Holden,
288
M.T., Zhang, C., Jiang, X., Koizumi, N., Taylor, K., Galloway, R., Hoffmaster, A.R.,
289
Craig, S., Smythe, L.D., Hartskeerl, R.A., Day, N.P., Chantratita, N., Feil, E.J.,
290
Aanensen, D.M., Spratt, B.G., and S.J. Peacock, 2013: A single multilocus sequence
291
typing (MLST) scheme for seven pathogenic Leptospira species. PLoS Negl Trop
292
Dis. 7(1), e1954.
293
Chappel, R. J., M. Goris, M. F. Palmer, and R. A. Hartskeerl, 2004: Impact of proficiency
294
testing on results of the microscopic agglutination test for diagnosis of leptospirosis.
295
Journal of clinical microbiology 42, 5484-5488.
12
296
Delooz, L., M. Mori, T. Petitjean, J. Evrard, G. Czaplicki, and C. Saegerman, 2015: Congenital
297
jaundice in bovine aborted foetuses: an emerging syndrome in southern Belgium.
298
Transboundary and emerging diseases 62, 124-126.
299
Dom, P. P., F. Haesebrouck, R. Vandermeersch, J. Descamps, and K. Van Ommeslaeghe, 1991:
300
Prevalence of Leptospira interrogans serovar hardjo antibodies in milk in Belgian dairy
301
herds. The Veterinary quarterly 13, 118-120.
302
Ellis, W. A., and S. W. Michna, 1977: Bovine leptospirosis: experimental infection of pregnant
303
heifers with a strain belonging to the Hebdomadis serogroup. Research in veterinary
304
science 22, 229-236.
305
306
Ellis, W. A., J. J. O'Brien, S. D. Neill, and D. G. Bryson, 1986: Bovine leptospirosis:
experimental serovar hardjo infection. Veterinary microbiology 11, 293-299.
307
Evangelista, K. V., and J. Coburn, 2010: Leptospira as an emerging pathogen: a review of its
308
biology, pathogenesis and host immune responses. Future microbiology 5, 1413-1425.
309
Guerra, M. A., 2009: Leptospirosis. Journal of the American Veterinary Medical Association
310
234, 472-478, 430.
311
Hartman, E. G., P. Franken, B. A. Bokhout, and D. J. Peterse, 1989: [Leptospirosis in cattle;
312
milker's fever in cattle farmers]. Tijdschrift voor diergeneeskunde 114, 131-135.
313
Hartskeerl, R. A., and W. J. Terpstra, 1996: Leptospirosis in wild animals. The Veterinary
314
315
316
317
318
319
quarterly 18 Suppl 3, S149-150.
Herrmann, J. L., 1993: Genomic techniques for identification of Leptospira strains. Pathologiebiologie 41, 943-950.
Jolley, K. A., and M. C. Maiden, 2010: BIGSdb: Scalable analysis of bacterial genome variation
at the population level. BMC bioinformatics 11, 595.
Levett, P. N., 2001: Leptospirosis. Clinical microbiology reviews 14, 296-326.
13
320
Levett, P. N., 2004: Leptospirosis: a forgotten zoonosis? Clinical and Applied Immunology
321
Reviews 4, 435–448.
322
Levett, P. N., R. E. Morey, R. L. Galloway, D. E. Turner, A. G. Steigerwalt , and L. W. Mayer,
323
2005: Detection of pathogenic leptospires by real-time quantitative PCR. Journal of
324
Medical Microbiology 54(1): 45-49.
325
Levett, P. N., 2015: Systematics of Leptospiraceae p. 11-20. In Ben Adler. Leptospira and
326
Leptospirosis, Current Topics in Microbiology and Immunology. Springer Berlin
327
Heidelberg.
328
Lin, M., O. Surujballi, K. Nielsen, S. Nadin-Davis, and G. Randall, 1997: Identification of a
329
35-kilodalton serovar-cross-reactive flagellar protein, FlaB, from Leptospira
330
interrogans by N-terminal sequencing, gene cloning, and sequence analysis. Infection
331
and immunity 65, 4355-4359.
332
Mayer-Scholl, A., E. Luge, A. Draeger, K. Nockler, and B. Kohn, 2013: Distribution of
333
Leptospira serogroups in dogs from Berlin, Germany. Vector borne and zoonotic
334
diseases 13, 200-202.
335
Morey, R. E., R. L. Galloway, S. L. Bragg, A. G. Steigerwalt, L. W. Mayer, and P. N. Levett,
336
2006: Species-specific identification of Leptospiraceae by 16S rRNA gene sequencing.
337
Journal of clinical microbiology 44, 3510-3516.
338
Mori, M., M. Esbroeck, S. Depoorter, W. Decaluwe, S. J. Vandecasteele, D. Fretin, and M.
339
Reynders, 2015: Outbreak of leptospirosis during a scout camp in the Luxembourg
340
Belgian province, Belgium, summer 2012. Epidemiology and infection 143, 1761-1766.
341
OIE
Terrestrial
Manual,
2014:
Leptospirosis.
Available
atw:
342
http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/2.01.09_LEPTO.pdf
343
(accessed 19 November 2015).
14
344
Ryan, E. G., N. Leonard, L. O'Grady, M. L. Doherty, and S. J. More, 2012: Herd-level risk
345
factors associated with Leptospira Hardjo seroprevalence in Beef/Suckler herds in the
346
Republic of Ireland. Irish veterinary journal 65, 6.
347
Smith, C. R., M. R. McGowan, C. S. McClintock, B. G. Corney, P. J. Ketterer, L. Smythe, and
348
W. Ward, 1997: Experimental Leptospira borgpetersenii serovar hardjo infection of
349
pregnant cattle. Australian veterinary journal 75, 822-826.
350
Terpstra, W. J., 1992: Typing Leptospira from the perspective of a reference laboratory. Acta
351
Leidensia 60, 79-87.
352
Tables and figures caption
353
Table I. List of pathogens included and diagnostic methods applied in the standardized panel
354
of analyses
Fetus
Foetal serum
Maternal serum
Methods
Methods
Pathogens
Samples
Methods
Abomasal fluid
Culture
Campylobacter foetus spp. Abomasal fluid
Culture
Coxiella burnetii
Abomasal fluid
PCR**
Listeria monocytogenes
Abomasal fluid
Culture
Mycotic agents
Abomasal fluid
Culture
Opportunistic bacteria
Abomasal fluid
Culture $
Salmonella spp.
Abomasal fluid
Culture
BTV-8
Brain
PCR*
Neospora caninum
Brain
PCR
Schmallenberg virus
Brain
PCR*
BoHV-4
Spleen
PCR
ELISA Ab
BVDV
Spleen
ELISA Ag
ELISA Ab
Brucella spp.
SAW /ELISA Ab
ELISA Ab
ELISA Ab
ELISA Ab
15
Leptospira serovar Hardjo
ELISA Ab
355
356
Legend: PCR, Polymerase chain reaction; Ab, Antibody; Ag, Antigen; SAW, Sero-
357
agglutination of Wright; $, Only the presence of a pure culture on blood agar is indicative of
358
opportunistic bacteria; *, Applied only if suspected case (congenital abnormalities); BVDV,
359
Bovine Viral Diarrhoea Virus; BTV-8, Bluetongue virus serotype 8; BoHV-4, Bovine
360
herpesvirus 4.
361
16
362
363
364
Table II. Distribution of MAT results among the tested cow sera according to different leptospiral serogroup (only serogroups with positive
results are listed)
Titer
Grippotyphosa Australis
Negative
25
1/160
4
1/320
3
1/640
4
1/1280
6
Total positive 17
Total sera tested 42
365
29
2
2
5
4
13
42
IcteroAutumnalis Panama Pyrogenes Sejroe Cynopteri Hebdomadis
haemorrhagiae
36
4
2
6
42
35
2
3
1
1
7
42
37
2
3
5
42
40
1
1
2
42
39
2
39
3
41
1
1
3
42
3
42
1
42
366
Table III. Results of serological and antigenical analysis according to Leptospira serogroup
367
(only serogroups with positive results are listed, the highest titer of each serogroup is presented)
Leptospiral serogroup
ID
CI-11-023436
CI-11-029715
CI-12-000889
CI-12-001123
CI-13-006899
CI-13-011101
CI-13-018383
CI-13-019237
CI-13-022971
CI-13-032292
CI-13-032707
CI-14-034435
CI-14-034966
CI-14-037270
CI-14-038297
CI-14-038323
CI-14-038596
CI-14-040708
CI-14-042496
CI-14-042765
CI-14-044328
CI-14-044569
CI-14-044707
CI-14-046867
CI-14-047394
CI-14-047531
CI-14-047968
CI-14-048785
CI-14-049712
CI-14-050512
CI-14-050870
CI-14-057066
CI-14-057564
CI-14-057925
CI-14-058607
CI-14-058713
CI-14-061536
CI-14-062221
CI-14-063728
CI-14-065178
Pathogenic
Leptospira
strains (PCR)
Pos
Genotyping
(MLST)
L. interrogans
Pos
Pos
Neg
Pos
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
*
L. kirschneri
*
*
AUS AUT CYN GRI HEB ICT PAN PYR SEJ
640
160
1280
320
1280
160
640
1280
1280
640
640
320
-
- 640 320 - 160
- 320 - 640 - 160 - 160 1280 160 - 160 - 160 1280 - 160 640 - 320 - 160 - 160 - 640 160 - 320 - 1280
- 640 - 160 1280 - 1280 320 - 160 320 - 320 - 160 640 - 320 640 - 160
- 640 - 1280 - 1280 - 1280 - 160 - 160 -
CI-14-067483
CI-14-069253
368
Pos
Neg
640 320
-
-
320
-
-
-
-
-
369
Legend:
370
* Unsuccessful amplification and sequencing; AUS, Australis ; AUT, Autumnalis ; CYN,
371
Cynopteri ; GRI, Grippotyphosa; HEB, Hebdomadis; ICT, Icterohaemorrhagiae ; PAN, Panama
372
; PYR, Pyrogenes ; SEJ, Sejroe.
373
2
374
Figure captions
375
Figure 1. Geographical distribution of icteric abortion's case, years 2008-2014 (N=152)
376
Legend: White dots correspond to icteric cases where complementary analysis (MAT and/or
377
RT-PCR) are performed; black dots corresponds to icteric cases without complementary
378
analysis.
379
380
Figure 2. Trends of icteric bovine aborted fetuses rate and the absolute number of notified
381
abortions
382
3
383
Fig. 1
384
385
4
386
Fig. 2
387
388
389
5